Uses of agents that bind immune system components

ABSTRACT

The present invention relates to the use of one or more compounds that can inhibit immune system costimulation by OX40 in the preparation of pharmaceutical compositions to treat, ameliorate or prevent diseases associated with virus, bacteria or other infective agent.

This invention relates to uses of agents, such as biological productswhich can interact with immune system components, to treat or amelioratediseases; to materials and compositions useful for such purposes; and totheir preparation and the preparation of components thereof. Among theseuses are uses as adjuvants for vaccines directed against viruses.

OX40 (also known as ACT-4, CD134 and Tumour Necrosis Factor ReceptorSuperfamily member 5) is a cell surface receptor that can occur on thesurface of activated CD4⁺ T-cells. WO95/12673 describes human OX40, anddiscloses an isolated form of OX-40, as well as fragments of OX40 andbiologically-active derivatives of OX40, e.g. fusion proteins such as afusion protein comprising an OX40 extracellular domain sequence fusedwith the sequence of an IgG heavy-chain constant-region domain. It alsodiscloses methods of using binding agents related to OX40, e.g.antibodies that bind to OX40, to alter immune responses.

It has been proposed to abrogate harmful cell-mediatedimmunopathological responses by using OX40:Ig fusion protein (oranti-OX40 ligand antibodies) to block the interaction between the T celland antigen-presenting cell (APC). Using this approach, several authorshave reported improving colitis in SCID mice, inflammatory boweldisease, allergic encephalomyelitis, acute graft versus host disease andcollagen-induced arthritis.

Anti-inflammatory activity was reported in particular by Higgins et al(1999) J Immunol 162:486-93. Further information about OX40 and relatedmaterials is contained in this paper and in published literature citedtherein, all of which documents are hereby incorporated herein byreference to the fullest extent permitted by law.

It is one of the aims of the present invention to provide new treatmentsfor inflammatory disease conditions. Further aims and aspects of theinvention are indicated by the description given hereinbelow.

In one of its aspects, the invention provides the use of one or morecompounds that can inhibit immune system costimulation by OX40 in thepreparation of pharmaceutical compositions to treat, ameliorate orprevent diseases associated with virus, bacteria or other infectiveagent.

In another aspect, the invention provides a method for the treatment,amelioration or prevention of diseases associated with virus, bacteriaor other infective agent, comprising administering to a subject one ormore compounds that can inhibit immune system costimulation by OX40.

Viral infections suitable for treatment by the medicaments of thepresent invention include infection with influenza or RSV or relatedviruses (e.g. negative-stranded viruses with segmented genomes: forwhich see e.g. WO 91/03552). In particular embodiments, the invention isuseful in relation to prevention or reduction of lung inflammationcaused by influenza virus and/or by respiratory syncytial virus (RSV)and/or bacterial infection by e.g. C neoformans.

Illnesses induced by infection of the lung by RSV and influenza virusare considered to be caused by the recruitment of an excessiveinflammatory infiltrate. Reduction of this infiltrate by, for example,TNF depletion can eliminate weight loss, cachexia and immobility ininfected animals. During infection, both CD4⁺ and CD8⁺ cells arebelieved to be recruited, which secrete large amounts of IFN-gamma.

According to one aspect of the invention, it has been found thatinhibition of OX40 costimulation, e.g. by a fusion protein based on OX40and an immunoglobulin, preferably IgG, can prevent or amelioratevirus-induced immunopathology, e.g. pathology associated with infectionby influenza or RSV virus or related viruses, e.g. in the lung. Thefusion protein may comprise the extracellular domain of human OX40linked via its C-terminal to the N-terminal of the constant domain of ahuman immunoglobulin (preferably the heavy chain thereof, and morepreferably the hinge, CH2 and CH3 regions thereof). The humanimmunoglobulin may be IgGγ. Accordingly, substances that are able toinhibit OX40 costimulation can prevent or ameliorate such pathology, andcan be useful in pharmaceutical preparations for the treatment ormanagement of diseases in which such pathology is a feature.Additionally, it has been found that such a fusion protein canameliorate lung infection by C. neoformans.

The invention also provides a product containing one or more compoundsthat can inhibit immune system costimulation by OX40; and ananti-inflammatory drug as a combined preparation for simultaneous,sequential or separate use in treating, ameliorating or preventingdiseases associated with virus, bacteria or other infection.

In another aspect, the invention provides a composition comprising avaccine and one or more compounds that can inhibit immune systemcostimulation by OX40. The vaccine may be an anti-viral oranti-bacterial vaccine.

In a further aspect, OX40:IgG fusion proteins and biologics with similarbinding activity are provided as vaccine adjuvants for vaccines againstviruses and bacteria. The adjuvants can be given either at the same timeas or after the vaccines themselves. Vaccines against viruses, such asagainst influenza or RSV or related viruses, with which the adjuvantscan be used in combination or association, include conventional vaccinesdirected against such viruses. Generally the range of vaccines withwhich the adjuvants can be associated or combined includes killedvaccines, subunit vaccines and genetically disabled vaccines as forexample those disclosed in relation to influenza viruses and relatedviruses in WO 91/03552. Thus, the invention also provides a productcontaining an anti-viral vaccine and one or more compounds that caninhibit immune system costimulation by OX40 as a combined preparationfor simultaneous, sequential or separate use in vaccination against saidvirus.

Compounds that can inhibit immune system costimulation by OX40 includeproteins related to OX40 that have this ability. Examples of suchproteins are described in WO 95/12673. Thus, the compound may be humanOX40, the amino acid and nucleic acid sequences of which is shown inFIG. 1, as well as other proteins that represent allelic, nonallelic,and higher cognate variants of OX40, and natural or induced mutants ofany of these. Usually, OX40 polypeptides will also show substantialsequence identity with the sequence of FIG. 1. Typically, an OX40polypeptide will contain at least 4 and more commonly 5, 6, 7, 10 or 20,50 or more contiguous amino acids from the sequence of FIG. 1. It iswell known in the art that functional domains, such as binding domainsor epitopes can be formed from as few as four amino acids residues.

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotides: “reference sequence”, “comparisonwindow”, “sequence identity”, “percentage of sequence identity”, and“substantial identity”. A “reference sequence” is a defined sequenceused as a basis for a sequence comparison; a reference sequence may be asubset of a larger sequence, for example, as a segment of a full-lengthcDNA or gene sequence given in a sequence listing, such as apolynucleotide sequence shown in FIG. 1, or may comprise a complete cDNAor gene sequence. Generally, a reference sequence is at least 20nucleotides in length, frequently at least 25 nucleotides in length, andoften at least 50 nucleotides in length. Since two polynucleotides mayeach (1) comprise a sequence (i.e., a portion of the completepolynucleotide sequence) that is similar between the twopolynucleotides, and (2) may further comprise a sequence that isdivergent between the two polynucleotides, sequence comparisons betweentwo (or more) polynucleotides are typically performed by comparingsequences of the two polynucleotides over a “comparison window” toidentify and compare local regions of sequence similarity. A “comparisonwindow”, as used herein, refers to a conceptual segment of at least 20contiguous nucleotide positions wherein a polynucleotide sequence may becompared to a reference sequence of at least 20 contiguous nucleotidesand wherein the portion of the polynucleotide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) of 20% or lessas compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. Optimalalignment of sequences for aligning a comparison window may be conductedby the local homology algorithm of Smith & Waterman, Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Natl. Acad. Sci. (USA) 85:2444 (1988), by computerizedimplementations of these algorithms (FASTDB (Intelligenetics), BLAST(National Center for Biomedical Information) or GAP, BESTFIT, FASTA, andTFASTA (Wisconsin Genetics Software Package Release 7.0, GeneticsComputer Group, 575 Science Dr., Madison, Wis.)), or by inspection, andthe best alignment (i.e., resulting in the highest percentage ofsequence similarity over the comparison window) generated by the variousmethods is selected. The term “sequence identity” means that twopolynucleotide sequences are identical (i.e., on anucleotide-by-nucleotide basis) over the window of comparison. The term“percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, U, or I) occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the window of comparison (i.e., the window size),and multiplying the result by 100 to yield the percentage of sequenceidentity. The terms “substantial identity” as used herein denotes acharacteristic of a polynucleotide sequence, wherein the polynucleotidecomprises a sequence that has at least 70, 80 or 85% sequence identity,preferably at least 90 to 95% sequence identity, more usually at least99% sequence identity as compared to a reference sequence over acomparison window of at least 20 nucleotide positions, frequently over awindow of at least 25-50 nucleotides, wherein the percentage of sequenceidentity is calculated by comparing the reference sequence to thepolynucleotide sequence which may include deletions or additions whichtotal 20 percent or less of the reference sequence over the window ofcomparison.

As applied to polypeptides, the term “substantial identity” means thattwo peptide sequences, when optimally aligned, such as by the programsBLAZE (Intelligenetics) GAP or BESTFIT using default gap weights, shareat least 70% or 80% sequence identity, preferably at least 90% sequenceidentity, more preferably at least 95% sequence identity or more (e.g.,99% sequence identity). Preferably, residue positions which are notidentical differ by conservative amino acid substitutions. Conservativeamino acid substitutions refer to the interchangeability of residueshaving similar side chains. For example, a group of amino acids havingaliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulphur-containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine, andasparagine-glutamine.

The term “higher cognate variants” as used herein refers to a genesequence that is evolutionarily and functionally related between humansand higher mammalian species, such as primates, porcines and bovines.The term does not include gene sequences from rodents, such as rats.

OX40 polypeptides will typically exhibit substantial amino acid sequenceidentity with the amino acid sequence of FIG. 1, and be encoded bynucleotide sequences that exhibit substantial sequence identity with thenucleotide sequence of FIG. 1. Such nucleotides will also typicallyhybridise to the sequence of FIG. 1 under stringent conditions.Stringent conditions are sequence dependent and will be different indifferent circumstances. Generally, stringent conditions are selected tobe about 5° C. lower than the thermal melting point (Tm) for thespecific sequence at a defined ionic strength and pH. The Tm is thetemperature (under defined ionic strength and pH at which 50% of thetarget sequence hybridises to a perfectly matched probe. Typically,stringent conditions will be those in which the salt concentration is atleast about 0.02 molar at pH 7 and the temperature is at least about 60°C. As other factors may significantly affect the stringency ofhybridisation, including, among others, base composition and size of thecomplementary strands, the presence of organic solvents and the extentof base mismatching, the combination of parameters is more importantthan the absolute measure of any one.

Usually, OX40 polypeptides will share at least one antigenic determinantin common with a protein having the sequence of FIG. 1. The existence ofa common antigenic determinant is evidenced by cross-reactivity of thevariant protein with any antibody prepared against a protein having thesequence of FIG. 1. Cross-reactivity is often tested using polyclonalsera against a protein having the sequence of FIG. 1, but can also betested using one or more monoclonal antibodies against a protein havingthe sequence of FIG. 1, such as the antibody designated L106 which isproduced by the hybridoma line HBL106, deposited as ATCC HB 11483, (atthe American Type Culture Collection, 12301 Parklawn Drive, Rockville,Md. 20852, on 3 Nov. 1993).

Polypeptides useful in the present invention may contain modifiedpolypeptide backbones. Modifications include chemical derivatisations ofpolypeptides, such as acetylations, carboxylations and the like. Theyalso include glycosylation modifications (N- and O-linked) andprocessing variants of a typical polypeptide. T

The amino acid sequence shown in FIG. 1 contains a 22 or 24 amino acidputative N-terminal signal sequence. It also contains a singleadditional hydrophobic stretch of 27 amino acids spanning residues213-240. The hydrophobic stretch probably corresponds to a transmembranedomain. The 189 or 191 amino acids (depending on the exact location ofthe signal cleavage site) of the FIG. 1 sequence amino-proximal to thetransmembrane segment are designated the extracellular domain, while the37 amino acids carboxy-proximal to the transmembrane segment aredesignated the intracellular domain. From the amino-terminus, theextracellular domain has an amino-terminal hydrophobic putative signalsequence, and three intrachain loops formed by disulphide bondingbetween paired cysteine residues.

Although not all of the domains discussed above are necessarily presentin proteins useful in the invention, an extracellular domain is expectedto be present in most. Indeed, in some proteins, only an extracellulardomain is present, and the natural state of such proteins is not ascell-surface bound proteins, but as soluble proteins, for example,dispersed in an extracellular body fluid.

Besides substantially full-length polypeptides, biologically-activefragments of the polypeptides are useful in the present invention.Significant biological activities include antagonism of the binding ofan OX40 polypeptide to its ligand. A segment of an OX40 protein or adomain thereof will ordinarily comprise at least about 5, 7, 9, 11, 13,16, 20, 40, or 100 contiguous amino acids. Some fragments will containonly extracellular domains, such as one or more disulphide-bonded loops.Such fragments will often retain the binding specificity of an intactOX40 polypeptide, but will be soluble rather than membrane bound.

Fragments or analogues comprising substantially one or more functionaldomain (e.g., an extracellular domain) of OX40 and related peptides canbe fused to heterologous polypeptide sequences, such that the resultantfusion protein exhibits the functional property(ies) conferred by theOX40 receptor fragment and/or the fusion partner.

Recombinant globulins (Rg) formed by fusion of OX40 fragments andimmunoglobulin components often have most or all of the physiologicalproperties associated with the constant region of the particularimmunoglobulin class used. For example, the recombinant globulins may becapable of fixing complement, mediating antibody dependent celltoxicity, stimulating B cells, or traversing blood vessel walls andentering the interstitial space. The recombinant globulins are usuallyformed by fusing the C-terminus of an OX40 extracellular domain to theN-terminus of the constant region domain of a heavy chainimmunoglobulin, thereby simulating the conformation of an authenticimmunoglobulin chain. The immunoglobulin chain is preferably of humanorigin. Recombinant globulins are usually soluble and may have aprolonged serum half-life, the capacity to lyse target cells for whichOX40 receptor has affinity, by effector functions, and the capacity tobind molecules such as protein A and G, which can be used to immobilisethe recombinant globulin in binding analyses.

Also suitable for use in the present invention are antibodies which bindto OX40. Such antibodies can be produced by a variety of means. Theproduction of non-human monoclonal antibodies, e.g., murine, rat and soforth, is well known. Several techniques for generation of humanmonoclonal antibodies have also been described, see, e.g., U.S. Pat. No.5,001,065. One technique that has successfully been used to generatehuman monoclonal antibodies against a variety of antigens is the triomamethodology of Ostberg et al. (1983), Hybridoma 2:361-367, U.S. Pat. No.4,634,664, and U.S. Pat. No. 4,634,666. The antibody-producing celllines obtained by this method are called triomas, because they aredescended from three cells: two human and one mouse. Triomas have beenfound to produce antibody more stably than ordinary hybridomas made fromhuman cells. An alternative approach is the generation of humanisedimmunoglobulins by linking the CDR regions of non-human antibodies tohuman constant regions by recombinant DNA techniques (Queen et al.,Proc. Natl. Acad. Sci. USA 86:10029-10033 (1989) and WO 90/07861). Thehumanised immunoglobulins have variable region framework residuessubstantially from a human immunoglobulin (termed an acceptorimmunoglobulin) and complementarity determining regions substantiallyfrom a mouse immunoglobulin, e.g., the L106 antibody (referred to as thedonor immunoglobulin). The constant region(s), if present, are alsosubstantially from a human immunoglobulin. The human variable domainsare usually chosen from human antibodies whose framework sequencesexhibit a high degree of sequence identity with the murine variableregion domains from which the CDRs were derived. The heavy and lightchain variable region framework residues can be derived from the same ordifferent human antibody sequences. The human antibody sequences can bethe sequences of naturally occurring human antibodies or can beconsensus sequences of several human antibodies (WO 92/22653). Certainamino acids from the human variable region framework residues areselected for substitution based on their possible influence on CDRconformation and/or binding to antigen. Investigation of such possibleinfluences is by modelling, examination of the characteristics of theamino acids at particular locations, or empirical observation of theeffects of substitution or mutagenesis of particular amino acids.

A further approach for isolating DNA sequences which encode a humanmonoclonal antibody or a binding fragment thereof is by screening a DNAlibrary from human B cells according to the general protocol outlined byHuse et al., Science 246:1275-1281 (1989) and then cloning andamplifying the sequences which encode the antibody (or binding fragment)of the desired specificity. The protocol described by Huse is renderedmore efficient in combination with phage display technology. See, e.g.,WO 91/17271 and WO 92/01047. Phage display technology can also be usedto mutagenise CDR regions of antibodies previously shown to haveaffinity for ACT-4 receptors or their ligands. Antibodies havingimproved binding affinity are selected.

Anti-OX40 antibodies that specifically bind to the same epitope as theL106 antibody are usually identified by a competitive binding assay. Theassay has three components, an OX40 polypeptide, L106 antibody, which isusually labelled, and the antibody under test. The test antibody bindsto the same epitope as the L106 antibody if it reduces the amount ofL106 antibody that specifically binds to OX40. Antibodies binding to thesame epitope as L106 may exhibit a substantially, but not completely,identical amino acid sequence to the L106 antibody, or may have anunrelated primary structure to the L106 antibody.

Anti-OX40 antibodies having a different binding specificity than L106(i.e., which bind to a different epitope) are identified by acomplementary approach. Test antibodies are screened for failure tocompete with the L106 antibody for binding to an OX40 polypeptide. Theextent of screening can be reduced by generating antibodies with aprotocol in which a fragment lacking a specific epitope bound by L106 isused as an immunogen.

Fragments of such antibodies may be used. Antibody fragments includeseparate heavy chains, light chains Fab, Fab′ F(ab′)², Fabc, and Fv.Fragments are produced by recombinant DNA techniques, or by enzymic orchemical separation of intact immunoglobulins.

Other agents suitable for use in the invention may be found by screeningfor OX40 agonists. Compounds that block OX40-induced DNA synthesis orprotein phosphorylation are antagonists. Antagonistic activity can alsobe determined from other functional or physical endpoints of leukocyteactivation, or from clinically desirable or undesirable outcomes, suchas cytolytic activity, or extravasation of leukocytes into tissues fromblood vessels.

The ability of agents to antagonise T-cell proliferation in vitro can becorrelated with the ability to affect the immune response in vivo. Invivo activity is typically assayed using suitable animal models such asmice or rats.

The form of medicament of the invention depends on the intended mode ofadministration. The compositions may also include, depending on theformulation desired, pharmaceutically-acceptable, non-toxic carriers ordiluents, which are defined as vehicles commonly used to formulatepharmaceutical compositions for animal or human administration. Thediluent is selected so as not to affect the biological activity of thecombination. Examples of such diluents are distilled water,physiological saline, Ringer's solutions, dextrose solution, and Hank'ssolution. In addition, the pharmaceutical composition or formulation mayalso include other carriers, adjuvants, or nontoxic, nontherapeutic,nonimmunogenic stabilizers and the like.

Amounts effective for use in the present invention will depend upon theseverity of the condition, the general state of the patient, and theroute of administration, and combination with other immunosuppressivedrugs, if any, but generally range from about 10 ng to about 1 g ofactive agent per dose, with single dosage units of from 10 mg to 100 mgper kg patient being commonly used. Pharmaceutical compositions can beadministered systemically by intravenous infusion, or locally byinjection.

In prophylactic applications, pharmaceutical compositions areadministered to a patients at risk of, but not already suffering anundesired immune reaction. The amount of antibody to be administered isa “prophylactically effective dose,” the precise amounts of which willdepend upon the patient's state of health and general level of immunity,but generally range from 10 ng to 1 g per dose, especially 10 mg to 100mg per patient.

The therapeutic agents of the present invention can also be combinedwith traditional therapeutics, and can be used to lower the dose of suchagents to levels below those associated with side effects. For example,other immunosuppressive agents such as antibodies to the α3 domain, Tcell antigens (e.g., OKT4 and OKT3), antithymocyte globulin, as well aschemotherapeutic agents such as cyclosporine, glucocorticoids,azathioprine, prednisone can be used in the present invention.

A pharmaceutical composition used in the present connection fortreatment of an (human or non-human) animal, preferably mammal, forexample to reduce lung inflammation associated with infection by RSV orinfluenza virus or viruses related thereto, is for example a sterileinjectable aqueous formulation comprising an OX40:IgG fusion proteinwherein the origin of the sequences of the OX40 and the IgG in thefusion protein is from the same (human or non-human) animal species asthe species to be treated, in an amount in for example the range ofabout 10 μg to about 500 μg, e.g. of the order of about 100 μg. Suchdosages have produced effects as described for example in theexperimental report below, and dosages of these and other alternativebinding agents that can be used in accordance with the present inventioncan be determined by dose optimisation to achieve these or other desiredlevels of activity.

Preferred features of each aspect of the invention are as for each ofthe other aspects mutatis mutandis. The prior art documents mentionedherein are incorporated to the fullest extent permitted by law.

The invention will be described further in the following non-limitingexample. Reference is made to the accompanying drawings in which:

FIG. 1 shows the amino acid and nucleic acid sequences of human OX40;

FIG. 2 is a graph illustrating the mean % of original weight afterinfection for influenza-infected mice when treated with OX40:Ig (opensymbols) or with PBS (closed symbols);

FIG. 3 is a graph illustrating cumulative illness (scored using anestablished grading system based on degree of cachexia and mobility)after infection for OX40:Ig treated mice (open bars) and control mice(closed bars);

FIG. 4 is a graph illustrating total numbers of CD4⁺ and CD8⁺ T cellsinfiltrated into bronchoalveolar lavage (BAL) during influenza infectionfor OX40:Ig treated mice (open symbols) and with control animals (closedsymbols);

FIG. 5 is a graph illustrating the mean % of original weight afterinfection for RSV-infected mice when treated with OX40:Ig (open symbols)or with PBS (closed symbols);

FIG. 6 is a graph illustrating total numbers of lymphocytes CD4⁺ andCD8⁺ T cells infiltrated into bronchoalveolar lavage (BAL) during RSVinfection for OX40:Ig treated mice (open symbols) and with controlanimals (closed symbols);

FIG. 7: OX40 was up-regulated on CD4⁺ and CD8⁺ T cells during infection.

Cells from the lungs of mock (solid histogram) or influenza (a,b), RSV(c,d) or C. neoformans (d,e) (dashed line) infected mice were stainedwith anti-CD4, -CD8 and -OX40 antibodies for 30 minutes on ice and thenfixed. Expression was analysed by flow cytometry collecting data on30,000 lymphocytes. The level of expression of OX40 on cells from mockinfected mice was indistinguishable from the isotype matched controlstained sample which has been omitted for clarity. These plots arerepresentative of 5 mice per group in two individual experiments;

FIG. 8: OX40L:Ig enhanced fungal clearance, reduced pulmonaryeosinophils and enhanced CD4⁺ T cells producing IFNγ.

C57B116 mice (n=5) were infected with C. neoformans and treated with PBS(▪) or OX40L:Ig (□) on days 0, 2, 5 and 7 of infection. 12 days afterinfection the number of cfu/lung was determined from lung homogenates(a). BAL eosinophils were enumerated in H and E stained cytocentrifugepreparations, each point representing an individual mouse (b). Thefrequency of IFN-γ (c) and IL-5 (d) producing cells in the lung wasassessed by ELISPOT assay (mean and standard deviation of 5 mice pergroup). IFNγ in CD4⁺ T cells was evaluated by intracellular cytokinestaining in Cryptococcus infected control (e) and OX40L:Ig (f) treatedmice. These results are representative of two experiments containing 5mice per group;

FIG. 9: OX40:Ig treatment prevented illness and over exuberant immuneresponses in RSV and influenza infected BALB/c mice.

Two groups of 4 mice were infected with influenza virus X31, one group(□) was treated with OX40:Ig on days 0, 2 and 5 of infection. Weightloss was monitored daily and expressed as a percentage of original bodyweight (a). Illness was scored from 1-5 based on the degree ofimmobility, cachexia and ruffled fur. The cumulative illness score wascalculated by treatment group (b). CD4⁺ (c and d) and CD8⁺ (e and f) Tcells in the airways of influenza (c and e) and RSV (d and f) infectedmice were determined by multiplying the percent positive cells by flowcytometry with the total viable cells recovered from the BAL;

FIG. 10: OX40:Ig enhanced virus specific antibodies and Th2 cytokinesbut reduced IFNγ in RSV infected mice treated with OX40:Ig. BALB/c micewere infected with RSV, treated with or without OX40:Ig fusion proteinand 7 days later serum and lung cells were removed. RSV titre in thelung was determined by plaque assay of homogenates from control (opencircles) or OX40:Ig treated (closed circles) mice (a). RSV specificantibody in the serum of control (▪) or OX40:Ig treated (□) mice wasdetermined by ELISA; each point representing an individual mouse (b).The frequency of lung cells secreting IFNγ (c and e) and IL-4 (d and f)in lung cell suspensions from RSV (c and d) or influenza virus (e and f)infected mice was determined by ELISPOT assay. The results represent themean and standard deviation of 5 mice per group. Open and filled barsrepresent OX40:Ig and control treated mice, respectively;

FIG. 11: OX40:Ig treatment reduced TNF and cellular infiltration intothe lung. Influenza virus infected mice were treated with PBS (a and c)or OX40:Ig (b and d). 7 days later lung cells were removed and stainedfor CD8 and intracellular TNF. Figures a and b show representative dotplots of airway CD8⁺ T cells (y axis) versus TNF (x axis). In someexperiments the lungs from PBS (c) or OX40:Ig (d) treated mice wereinflated and fixed with 2% formaldehyde, paraffin embedded and sectionsstained with H and E. These sections are representative of 5 mice pergroup in 3 independent experiments;

FIG. 12: Delayed OX40:Ig treatment reversed influenza virus inducedweight loss. Mice (n=5) were infected with influenza virus (X31) on dayzero and treated with PBS (▪) or OX40:Ig (□) on days 3, 5 and 6 ofinfection. Percent weight loss is calculated from the day of treatment,each line representing and individual mouse.

FIG. 13: OX40:Ig did not alter recall responses to influenza and RSVfollowing re-challenge. BALB/c mice (n=4) were infected with eitherinfluenza virus (a, c and e) or RSV (b, d and f) and treated with PBS(▪) or OX40:Ig (□). 3 weeks later, mice were challenged with homologousvirus and weight loss (a and b), CD4⁺ T cell (c and d) and CD8⁺ T cell(e and f) infiltrate 5 or 6 days post-infection was measured;

FIG. 14: OX40L:Ig treatment did not enhance RSV induced illness andpromoted virus-specific IFNγ and IL-4 responses. Mice were infected withRSV and treated with OX40L:Ig. The frequency of RSV specific lung Tcells producing IL-4 (a) and IFNγ (b) was determined by ELISPOT. Theresults represent individual control (▪) or OX40L:Ig (□) treated mice.RSV titres were determined in lung homogenates from control (closedsymbols) or OX40L:Ig (open symbols) treated mice (c);

FIG. 15: Time-course of C. neoformans infection is accompanied by OX40expression by CD4⁺ T cells in the lung and airways.

The number of cfu/lung was determined by plating out lung homogenate in10-fold serial dilutions on sabouraud dextrose, and BAL eosinophils wereenumerated as granulocytes by flow cytometry and confirmed using H and Estained cytocentrifuge preparations over a 45-day time-course experiment(a). Cells from the lungs of C. neoformans infected mice taken on days0,2,7,11,14,19 and 45 days post-infection were stained with anti-CD4 andanti-CD8 antibody and analysed by flow cytometry. Total numbers weredetermined by multiplying the percent positive cells by flow cytometrywith the total viable cells recovered from the lung (b). OX40 expressionby CD4⁺ T cells in the BAL and lung was analysed by flow cytometry (c).A representative plot of CD4 (y axis) versus OX40 (x axis) is shown inpanel d and is representative of 4-5 mice per group in two individualexperiments;

FIG. 16: OX40L:Ig enhances CD4⁺ T cell numbers in the lung during C.neoformans infection.

C57BL/6 mice (n=4) were infected with C. neoformans and treated withcontrol Ig (●) or OX40L:Ig (◯) on days 0, 2, 5, 8, and 11 afterinfection. Lung cells were taken on days 0,2,7,11,14, and 19post-infection and stained with anti-CD4 antibody. Total numbers weredetermined by multiplying the percent positive cells by flow cytometrywith the total viable cells recovered from the lung (a). **, p<0.005 forcontrol Ig vs OX40L:Ig. 11 days after infection, lungs from mice treatedwith either control Ig (b) or OX40L:Ig (c) were inflation fixed with 10%formaldehyde, paraffin embedded and sections stained with H and E. Thesesections are representative of 5 mice per group in 3 independentexperiments;

FIG. 17: OX40L:Ig enhances cellular proliferation in the lung Cellproliferation in the lungs of C. neoformans infected mice treated withisotype control Ig (a) and OX40L:Ig (b) was analysed by injection ofBrdU 90 minutes before sacrifice and stained with anti-BrdU monoclonalantibody. Proliferation of cells in the lungs was obtained by countingproportion of positively stained within perivascular infiltrates with atleast 500 cells counted per section (c);

FIG. 18: OX40L:Ig administration increases pathogen clearance andreduces associated pulmonary eosinophilia during C. neoformansinfection.

C. neoformans infected mice were treated with either control Ig (●) orOX40L:Ig (◯) days 0, 2, 5, 8, 11 and 14 days post-infection. At multipletime-points (days 0, 2, 7, 11, 14, 19), cfu/lung was determined fromlung homogenate (a) and BAL eosinophils were enumerated in H and Estained cytocentrifuge preparations (b). *, p<0.05 and **, p<0.005 forcontrol Ig vs OX40L:Ig;

FIG. 19: Enhanced OX40 signalling promotes IFN-γ production by CD4⁺ Tcells in the lung during C. neoformans infection.

IFN-γ production by CD4⁺ T cells 12 days post-infection was evaluated byintracellular cytokine staining in Cryptococcus infected control (a) andOX40L:Ig (b) treated mice. These results are representative of threeexperiments containing 5 mice per group. The ratio of IFN-γ/IL-5producing CD4⁺ T cells was calculated by dividing percentage ofIFN-γ-expressing CD4⁺ T cells by the percentage producing IL-5, asdetected by intracellular cytokine staining (c). **, p<0.005 for controlIg vs OX40L:Ig; and

FIG. 20: IFN-γ and IL-12 mediate OX40L:Ig-dependent inhibition ofeosinophilia and promotion of C. neoformans clearance but not CD4⁺ Tcell activation.

Groups of 5 wild type (C57BL/6), IFNγ^(−/−) and IL12^(−/−) mice (C57BL/6background) were infected with C. neoformans and treated with eitherOX40L:Ig or control antibody on days 0,2,5,8. Mice were sacrificed 12days post infection. CD4⁺ T cells (a) in the lung of infected mice weredetermined by multiplying the percent positive cells by flow cytometrywith the total viable cells recovered from the lung. Eosinophils wereenumerated from H and E stained cytocentrifuge preparations of BAL fluid(b). The cfu/lung were measured from lung homogenate (c). All resultsrepresent mean values of 5 mice per group. *, p<0.05 and **, p<0.005 forcontrol Ig vs OX40L:Ig in either wild type or IFN-γ^(−/−) mice.

EXAMPLES Example 1

Mice were infected separately with influenza virus, with RSV, or with acontrol infectious agent, Cryptococcus neoformans, which is anencapsulated yeast that causes lung eosinophilia in immuno-compromisedpatients. Unlike the viral infections, it is the replication ofCryptococcus that causes illness.

One group of mice was treated with OX40:Ig (which can bind the OX40Lligand naturally expressed on antigen presenting cells and prevent Tcell co-stimulation); the control group was treated withphosphate-buffered saline (PBS). Treatment was performed on the day ofinfection and then on alternate days until the experiment was terminatedand the animals killed. Weight loss and appearance were monitoredthroughout. At sacrifice, lung lavage, residual lung tissue, mediastinallymph nodes and serum were removed.

Influenza Virus Infection:

Weights

FIG. 2 shows that the weight loss induced by influenza is inhibited inmice treated with OX40:Ig (open symbols) compared with PBS-treated mice(closed symbols).

Illness Severity

FIG. 3 illustrates that a remarkable beneficial effect of OX40:Igadministration was also shown when the illness was scored using anestablished grading system based on degree of cachexia and mobility.OX40:Ig (open bars) treatment almost eradicates influenza-inducedillness seen in control animals (closed bars).

Cellular Infiltration

FIG. 4 illustrates that OX40:Ig treatment (open symbols) inhibitedcellular infiltration into the bronchoalveolar lavage (BAL) duringinfluenza infection. A reduction in total cell numbers and totallymphocytes (data not shown), together with total CD4⁺ and CD8⁺ T cellswas observed in treated groups compared with control animals (closedsymbols). A reduction in TNF-producing CD8⁺ T cells in the lung was alsoobserved following OX40:Ig administration. TNF has been implicated as acausative agent in viral-induced weight loss: therefore reduced levelsof this in OX40:Ig-treated animals explains inhibition of weight loss.

RSV Infection:

Weight Loss and Illness

In comparison to influenza, respiratory syncytial virus (RSV) is arelatively mild respiratory infection. BALB/c mice experience mildweight loss and illness during the first 6-8 days of infection.

FIG. 5 illustrates how OX40:Ig treatment reduced both weight loss andillness severity at later time-points (see below), although due to themild nature of RSV infections, major differences between control animals(closed symbols) and OX40:Ig-treated mice (open symbols) were less thanthose during influenza infection.

Cellular Infiltration

FIG. 6 illustrates that, as observed in influenza-inducedimmunopathology, OX40:Ig treatment (open symbols) reduced cellularinfiltration of lymphocytes, CD4⁺ and CD8⁺ T cells into the BAL during aprimary RSV infection.

Antibody Production

RSV also induces protective humoral immune responses during infection.Most important for vaccine development is an observation that OX40:Igadministration has led to an increase in total anti-RSV IgG levels inthe serum of RSV-infected mice (data not shown).

Accordingly, in another aspect of the invention OX40:IgG fusion proteinsand biologics with similar binding activity are provided (e.g. indosages as disclosed above), as vaccine adjuvants for vaccines againstviruses, for example against influenza or RSV or related viruses (e.g.negative-stranded viruses with segmented genomes: for which see e.g. WO91/03552). The adjuvants can be given either at the same time as orafter the vaccines themselves. Vaccines against viruses, such as againstinfluenza or RSV or related viruses, with which the adjuvants can beused in combination or association, include conventional vaccinesdirected against such viruses. Generally the range of vaccines withwhich the adjuvants can be associated or combined includes killedvaccines, subunit vaccines and genetically disabled vaccines as forexample those disclosed in relation to influenza viruses and relatedviruses in WO 91/03552.

Cryptococcus Neoformans Infection (Comparison)

In a murine model using BL/6 mice, Cryptococcus neoformans induces alarge eosinophilic infiltration into the lung. OX40:Ig treatment did notaffect these levels. During a primary Cryptococcus infection, miceexperience little or no weight loss. Therefore, extent of weightincrease was measured. OX40:Ig treatment actually appeared to induceslight weight loss compared with control mice which gained weight.

Cellular infiltration into the BAL during infection was reduced,although the extent of this varied between experiments. OX40:Igtreatment was also shown not to significantly affect clearance ofCryptococcus from the lung, spleen or brain compared with control mice.

Respiratory syncytial and influenza viruses cause acute respiratoryinfections and are cleared with relative ease by the immune system. Yet,the immunopathological results of these infections can be very serious.The disclosure above indicates that inhibition of OX40-mediated T cellcostimulation can reduce these inflammatory responses. OX40:Ig treatmentcan also induce an increased anti-RSV antibody response, probably due toOX40:Ig binding to OX40L on B cells, inducing IgG secretion (althoughthe inventors do not wish to be bound by this hypothesis).

Therefore, these results support that OX40:Ig has therapeutic usefulnessin the treatment of viral-induced inflammatory responses and inpromotion of protective antibody responses against a viral pathogen.

Example 2

Methods

Mice and Pathogens

8-12 week old female BALB/c and C57BL/6 mice (Harlan Olac Ltd, Bicester,UK) were kept in pathogen-free conditions according to Home Officeguidelines. Influenza A strain X31 (hemagglutinin [HA] titre 1024) was akind gift from Dr Alan Douglas (National Institute for Medical Research,London, UK). RSV (A2 strain) was grown in HEp-2 cells and assayed forinfectivity as described is Bangham,et al, J. Virol. 56, 55-59 (1985).C. neoformans strain 52, was obtained from the American Type CultureCollection (Rockville, USA) and for infection grown to stationary phase(48-72 hours) at room temperature on a shaker in Sabouraud dextrosebroth (1% neopeptone and 2% dextrose; Difco, Detroit, Mich.). Thecultures were washed in saline, counted on a haemocytometer and dilutedin sterile nonpyrogenic saline to the required infective dose.

Production of OX40 and OX40 Ligand Fusion Proteins

Murine OX40:mIgG1 fusion protein (OX40:Ig) was constructed by using achimeric cDNA that contained the extracellular domain of OX40 fused tothe constant region of murine IgG1. This construct was used to transfectclonal chinese hamster ovary cells and fusion protein was purified fromthe culture supernatant using protein G sepharose. The murineOX40L:murine IgG1 fusion protein (OX40L:Ig) was constructed as describedin Weinberg, et al. J. Immunol. 164, 2160-2169 (2000) using a chimericcDNA containing the C-terminal region of OX40L fused to the constantregion of murine IgG1.

Mouse Infections and Treatment

On day 0 BALB/c mice were anaesthetised and intranasally infected with1×10⁶ pfu RSV or 50 HA units influenza virus (in 50 μl). C. neoformansinfections (2×10⁴ pfu/mouse) were performed in C57BL/6 mice. Some groupsof mice were injected intraperitoneally (i.p.) with 100 μg OX40:Ig orOX40L:Ig fusion proteins (Xenova pharmaceuticals, Cambridge, UK) onvarious days after infection as indicated in the text. The weight ofmice and illness severity was monitored daily. Mice were sacrificed ondays 6 or 7 after viral infection and on day 12 after C. neoformansinfection by injection of 3 mg pentobarbitone and exsanguinated via thefemoral vessels. Bronchoalveolar lavage (BAL), lung and serum wereremoved using methods described in Hussell, et al, J. Gen. Virol. 77,2447-2455 (1996).

Quantification of Illness Severity

Illness severity was scored daily using the following criteria:0=healthy, 1=barely ruffled fur, 2=ruffled fur but active, 3=ruffled furand inactive, 4=ruffled fur, inactive and hunched, 5=dead.

Cell Recovery

BAL, lung tissue and serum were harvested by methods described inHussell, et al. J. Gen. Virol. 77, 2447-2455 (1996). Briefly, the lungsof each mouse were inflated 6 times with 1 ml 1 mM EDTA in EMEM andplaced in sterile tubes on ice. 100 μl BAL fluid from each mouse wascytocentrifuged onto glass slides. The remainder was centrifuged and thesupernatant removed and stored at −70° C. in 200 μl aliquots foranalysis of cytokines by ELISA. Cell viability was assessed using trypanblue exclusion and the pellet resuspended in RPMI containing 10% FCS, 2mM/ml L-glutamine, 50 U/ml penicillin and 50 μg/ml streptomycin (R10F)at a final concentration of 10⁶ cells/ml. Eosinophils were enumerated asgranulocytes by flow cytometry, using forward and side scatter.Identification was confirmed by counting eosinophils in H and E stainedcytocentrifuge preparations.

Flow Cytometric Analysis of Intracellular and Cell Surface Antigens

1×10⁶ BAL and lung derived cells were stained usingCy-chrome™-conjugated anti-CD4, anti-CD8-PE and either anti-CD45RB-FITC(all BD Pharmingen, Heidelberg, Germany) or anti-OX40-FITC (Serotec,Oxford, UK) for 30 min on ice. Cells were then fixed for 20 min at roomtemperature with 2% formaldehyde. To detect intracellular cytokines, 10⁶cells/ml were incubated with 50 ng/ml PMA (Sigma-Aldric, Poole, Dorset),500 ng/ml ionomycin (Calbiochem, Nottingham, UK), and 10 mg/ml brefeldinA (Sigma-Aldrich) for 4 hours at 37° C. Cells were then stained witheither anti-CD4-Cy-chrome™ or anti-CD8-Cy-chrome™ and fixed as describedabove. After permeabilization with PBS containing 1% saponin/1%BSA/0.05% azide (saponin buffer) for 10 min either FITC-conjugatedanti-TNF or anti-IFNγ (Pharmingen) and PE-conjugated anti-IL4(Pharmingen) diluted 1:50 in saponin buffer were added. 30 minutes latercells were washed once in saponin buffer and once in PBS/1% BSA/0.1%azide. All data was acquired on a FACSCalibur and analysed withCellQuest Pro software (BD Biosciences, Belgium).

Lung Histology

In some studies, lungs were inflated and fixed with 2% formalin in PBS,6-7 days after intranasal RSV or influenza virus and 12 days after C.neoformans infection. The inflated lungs were excised and embedded inparaffin wax by the histopathology department at the HammersmithHospital, UK. Thin sections were stained with haematoxylin and eosin (Hand E). The lungs from 4-5 mice per group were analysed.

Lung RSV Titer

Clearance of RSV was assessed in lung homogenates 2 and 4 days aftervirus challenge. After centrifugation at 4000 rpm for 4 minutes,supernatant was titrated in doubling dilutions on HEp-2 cell monolayersin flat bottomed 96 well plates. Twenty-four hours later, monolayerswere washed and incubated with peroxidase conjugated goat anti-RSVantibody (Biogenesis, Poole, UK). Infected cells were detected using3-amino-9-ethylcarbazole (AEC), infectious units being enumerated bylight microscopy.

Lung Influenza Virus Titer

The titer of influenza virus in the lungs was determined byhemagglutination assay. Briefly, lungs were removed, homogenized andcentrifuged to remove large cell debris. 50 μl supernatant was incubatedwith 1% turkey red blood cells and the hemagglutination titer determinedfrom the reciprocal of the highest dilution of virus showingagglutination (which represents 1 HAU/50 μl homogenate).

Enumeration of C. Neoformans from Lung Homogenates

Lungs were homogenised by passage through 100 μm cell strainers (BDlabware, New Jersey, USA). 100 μl of cell suspension was diluted in PBSand incubated at room temperature for 48 hours on sabouraud dextroseagar plates (Sigma). The total colony forming units per lung was thendetermined (number of colonies×dilution factor×original cell suspensionvolume).

RSV-Specific Antibody ELISA

Serum antibody was assessed by ELISA as described in Hussell, et al. EurJ Immunol 27, 3341-3349 (1997); Walzl, et al,. J.Exp.Med 193, 785-792(2001). ELISA antigen was prepared by infecting HEp-2 cells with RSVstrain A2 at 1 pfu/cell. When a significant cytopathic effect wasobserved the infected cells were harvested, centrifuged at 400 g,resuspended in 3 ml distilled water and then subjected to 2 minutes ofsonication (Ultrawave Ltd, Cardiff). 50 μl aliquots were stored at −20°C. until required. Microtitre plates were coated overnight with 100 μlof a 1/200 dilution of either sonicated RSV or HEp-2 cells alone. Afterblocking with 2% normal rabbit serum for 2 hours, dilutions of testsamples (diluted in PBS containing 1% HEp-2 lysate) were added for afurther hour at room temperature. Bound antibody was detected usingperoxidase-conjugated rabbit anti-mouse Ig and O-phenylene-diamine(Sigma) as a substrate. The reaction was stopped with 50 μl 2.5 Msulphuric acid. Optical densities were read at 490 nm. The amount of RSVspecific antibody was determined by subtracting the optical densityobtained by incubating serum on RSV coated plates from the same sampleincubated on HEp-2 coated plates.

Enzyme-Linked Immunospot (ELISPOT) for Murine Cytokines

ELISPOT assays were performed as described in Hussell, & Openshaw, J.Immunol. 165, 7109-7115 (2000). Sterile filter plates (multiscreenfilter plates, Millipore corporation, Bedford Mass., USA) were coatedovernight at 4° C. with 40 ug/ml rat anti-mouse IL-4, IL-5 or IFNγ(Pharmingen), washed and then blocked for 2 hours with 10% FCS/RPMI.Lung cells were added at 1×10⁶ cells/well with 4 doubling dilutions andcultured for 48 hours with either 1 pfu/cell RSV, 0.1 HA/cell influenzavirus X31, 5×10⁴ heat inactivated Cryptococcus or medium alone. Afterremoval of cells the site of cytokine production was detected using 1μg/ml biotinylated rat anti-mouse IL-4, IL-5 or IFNγ followed byalkaline phosphatase-labelled streptavidin using BCIP/NBT as thesubstrate. The frequency of cytokine producing cells was enumerated bylight microscopy.

Statistics

Statistical significance was evaluated using the student t test, 2tailed assuming unequal variance within the Minitab software program.

Results

Expression of OX40 on CD4⁺ and CD8⁺ T Cells During Lung Infection

Expression of OX40 on activated CD4⁺ T cells has previously beendescribed but expression by CD8⁺ T cells is unclear. Cells from thelungs of mice infected with influenza virus, RSV or C. neoformans wereanalysed for CD4, CD8 and OX40 expression using flow cytometry. Only lowlevels of OX40 were observed on lung cells from uninfected mice. Afterinfection with all pathogens however, OX40 expression increasedsignificantly on CD4⁺ T cells (FIG. 7 a, c and e). The level ofexpression was higher following influenza infection than in C.neoformans or RSV-infected mice. CD8⁺ T cells recruited in response toinfluenza infection also expressed high levels of OX40 (FIG. 7 b).Significant but low levels of OX40 were also observed on CD8⁺ T cellsfollowing RSV but not C. neoformans infection (FIG. 7 d, e).

OX40L:Ig Promotes IFNγ Production and Prevents Cryptococcus NeoformansInduced Lung Eosinophilia

Intranasal C. neoformans infection of C57BL/6 mice induces anon-protective Th2 cytokine response resulting in the accumulation of50-80% eosinophils in the lung. The OX40:Ig binds to OX40 on the T celland mimics APC derived co-stimulatory signals. C. neoformans could stillbe recovered from the lung of mock treated C57BL/6 mice 12 days afterinfection. Treatment with OX40L:Ig significantly reduced both the colonyforming units recovered (FIG. 8 a) and lung eosinophilia (FIG. 8 b).

This alteration in the pathological phenotype was accompanied by anincrease in the frequency of cells producing IFNγ (FIG. 8 c) whereas thefrequency of IL-5 producing cells was similar after OX40L:Ig treatment(FIG. 8 d). Intracellular cytokine staining revealed that both CD4⁺(FIGS. 2 e and f, p 0.019) and CD8⁺ (data not shown) T cells were thecellular source of IFNγ. Treatment with OX40L:Ig therefore enhanced IFNγproduction by T cells, reduced lung eosinophilia and promoted clearanceof C. neoformans from the lung. The converse treatment with OX40:Ig alsoreduced lung eosinophilia (from 1.57×10⁶+/−SD 0.44 to 0.99×10⁶+/−SD 0.3;p0.007) but not the pathogen burden. A reduction of CD4⁺ T cell numbersin the BAL (from 9.1×10⁴+/−SD 0.95 to 5.0×10⁴+/−SD 2.4; p0.02) was alsoobserved. In the case of OX40:Ig treatment eosinophilic responses wereabrogated because CD4⁺ T cells numbers were reduced.

OX40:Ig Treatment Prevented RSV and Influenza Virus Induced Illness andImmunopathology.

Lung infection of naïve BALB/c mice with RSV or influenza virus resultsin TNF and IFNγ driven weight loss and cachexia and is accompanied bythe recruitment of CD4⁺ and CD8⁺ T cells secreting type 1 cytokines. Inthis situation it is therefore desirable to reduce the type 1 cytokinephenotype. RSV and influenza infected mice were treated with OX40:Ig. Ininfluenza infected, control treated mice the experiment had to beterminated 6 days after infection due to severe weight loss. In aparallel group however OX40:Ig treatment abolished the weight loss (FIG.9 a) and illness severity. PBS treated influenza infected mice,especially at day 6, appeared hunched, immobile and severely cachexic;whereas OX40:Ig treated mice were indistinguishable from uninfectedcontrols (FIG. 9 b). A similar beneficial effect was also observedfollowing RSV infection (data not shown). During both infections OX40:Igtreatment significantly reduced the total cell recruitment, neutrophils,CD4⁺ (FIGS. 9 c and d) and CD8⁺ (FIGS. 9 e and f) T cells in the BAL.Mediastinal lymph nodes were also reduced in size in OX40:Ig treatedmice (from 14.0×10⁵+/−3.4 to 8.3×10⁵+/−SD 2.6, p 0.04).

The clearance of influenza and RSV (FIG. 10 a) was not affected byOX40:Ig treatment (data not shown). Though RSV titres were higher insome OX40:Ig treated mice this did not reach significance (p>0.05) andthe kinetics of RSV clearance was identical with PBS treated controls.All mice had cleared the virus by day 7. This may be due to the factthat innate immune mechanisms are very effective against lung viralinfections. During influenza infection in the BAL there were1.07×10⁴+/−SD 0.8 NK cells in mock treated mice compared to0.9×10⁴+/−0.3 in OX40:Ig treated mice. Similarly in the lung there were2.6×10⁴+/−SD 0.2 and 2.4+/−SD 0.4 NK cells in mock and OX40:Ig treatedmice, respectively. In addition virus specific antibody titres wereelevated in OX40:Ig treated mice (FIG. 10 b), which may be linked to thealteration in Th2 cytokines. Though there was no difference in thefrequency of cells producing IFNγ (FIGS. 10 c and e) a significantincrease was observed for IA (FIGS. 10 d and f) and IL-5 (data notshown) in OX40:Ig treated RSV and influenza infected mice. Byintracellular cytokine staining we also observed a decrease in theproportion of BAL CD8⁺ and CD8⁻ T cells producing TNF in OX40:Ig treatedmice (FIGS. 11 a and b). The absolute number of TNF-producing CD8⁺ Tcells was also significantly reduced in OX40:Ig treated mice. We havepreviously shown that TNF is intimately associated with illness andweight loss in both of these infections (Hussell, et al Eur J Immunol31, 2566-2573 (2001)). The reduction in TNF by OX40:Ig treatment maytherefore explain the beneficial effects observed in the current study.Treatment with anti-TNF antibodies also reduces total inflammatoryinfiltrate to the lung (Hussell, et al Eur J Immunol 31, 2566-2573(2001)); an effect clearly evident in H and E stained sections of lungtissue from OX40:Ig treated influenza infected mice. Cellularinfiltration around the airways and blood vessels was markedly reducedin OX40:Ig treated mice (FIGS. 11 c and d).

Delayed OX40:Ig Treatment Reduced the Immunopathology in InfluenzaInfected Mice with Established Illness.

For OX40:Ig treatment to be used in a clinical setting it must beeffective in patients with established disease. We therefore infectedBALB/c mice with influenza virus and administered OX40:Ig when they hadlost 20% of their starting weight. FIG. 12 shows that delayed treatmentreversed a decline in weight.

Recall Responses to Secondary Infections were not Altered by OX40:IgTreatment.

Since cellular recruitment was significantly reduced by OX40:Igtreatment during primary viral infections, we determined whether recallresponses were also affected by challenging mice with homologous virus 3weeks after the first infection. Influenza virus infected mice displayedless weight loss during a secondary challenge compared to a primaryinfection (compare weight loss profiles in FIGS. 3 a and 7 a). Treatmentwith OX40:Ig during the first infection did not alter the milder weightloss during re-challenge with either influenza (FIG. 13 a) or RSV (FIG.13 b). Similarly CD4⁺ and CD8⁺ T cell recruitment was unaltered inre-challenged mice following influenza (FIGS. 13 c and e) or RSV (FIGS.13 d and f) secondary infections.

OX40L:Ig Administration During RSV Enhanced RSV-Specific IFNγProduction.

The role of OX40 signalling in responses to RSV was further investigatedby administering OX40L:Ig to infected mice during lung infection (i.e.the converse treatment to OX40:Ig treatment). Surprisingly, weight losswas not affected (data not shown) despite enhanced levels ofRSV-specific IFNγ and IL-4 in 3/5 mice (FIG. 14). Clearance of RSV wasidentical to control treated mice (FIG. 14 c). It may therefore bepossible to promote anti-viral immunity to RSV using OX40L:Ig withoutenhancing illness.

Discussion

We show that 1) OX40 is up-regulated on both CD4⁺ and CD8⁺ T cells afterlung infection, 2) expression occurs in both Th1 and Th2 cytokineenvironments and, 3) manipulation of this single late co-stimulatorysignal alleviates the symptoms of three clinically important lunginfections. The effect on immunopathology is associated with a change inthe balance of Th1 and Th2 cytokines. OX40L:Ig administration enhancedIFNγ production in both C. neoformans and RSV infections: in the case ofC. neoformans, reducing harmful Th2-mediated pulmonary eosinophilia.Administration of OX40:Ig during C. neoformans infection also reducedeosinophilia by antagonizing CD4⁺ T cell numbers in the lung. Thebeneficial effect of OX40:Ig administration is not restricted to Th2induced immunopathology since Th1-mediated illness, weight loss and lunginflammatory cell infiltration during RSV or influenza virus infectionis also abrogated. Although OX40 expression by activated CD4⁺ T cellshas previously been described, few have reported expression by CD8⁺ Tcells (Al-Shamkhani, et al. Eur. J. Immunol. 26, 1695-1699 (1996)).Furthermore, the up-regulation of OX40 on CD8⁺ T cells is more dramaticduring influenza compared to RSV infection but the significance of thisis not known.

The use of fusion proteins provides a novel approach to studyco-stimulatory interactions in vivo compared with inhibitory anti-OX40antibodies, which though prevent T cell co-stimulation, also block thesignal to the APC via OX40L. With antibody treatment it is thereforedifficult to ascertain whether the observed effects are due to reduced Tcell activation or altered APC function since ligation of OX40L has beenshown to enhance maturation of human dendritic cells in vitro (Ohshima,et al, J. Immunol. 159, 3838-3848 (1997)) and impaired APC function hasbeen reported in OX40L^(−/−) mice (Murata, et al. J. Exp. Med. 191,365-374 (2000)). Stimulation of OX40L on B cells also increases Igsecretion (Stuber et al. Immunity. 2, 507-521 (1995); Morimoto, et al J.Immunol. 164, 4097-4104 (2000)), an effect observed in our studyfollowing OX40:Ig administration during RSV infection. This disadvantageof losing the signal to the T cell and APC is also relevant in OX40knockout mice, which have been used to study OX40 co-stimulation inasthma (Jember, et al J. Exp. Med. 193,387-392 (2001) and LCMV infectionKopf, et al Immunity. 11, 699-708 (1999). The role of OX40 innon-immunological processes is not known and so knockout mice may sufferfrom other developmental problems.

Although OX40:Ig administration during RSV or influenza infectionreduced lung T cell infiltration, the clearance of both viruses wasremarkably unaffected. The increase in virus specific antibody maycompensate for reduced cellular responses.

Alternatively, innate immune mechanisms, or the residual inflammatoryinfiltrate remaining in treated mice, may be enough to mediate virusclearance. Although NK cells play a pivotal role in immunity to RSVinfection (Hussell, & Openshaw, J. Immunol. 165, 7109-7115 (2000)),little is known of their function during influenza virus infection.Increased susceptibility to influenza in the senescence-acceleratedmouse is associated with impaired activity of NK cells, but CD8⁺ T cellsare also defective in this model (Dong, et al, J. Infect. Dis. 182,391-396 (2000)). NK cell responses in the current study were unalteredby OX40:Ig administration. RSV and particularly influenza virus arehighly inflammatory in the mouse. It is possible that a moderatereduction in the vigour of the inflammatory response may be beneficialwithout affecting virus clearance. Our results are consistent with theefficient control of Theiler's murine encephalomyelitis virus,Nippostrongylus brasiliensis, Leishmania major (Pippig, et al J.Immunol. 163, 6520-6529 (1999)) and influenza virus (Kopf, et al.Immunity. 11, 699-708 (1999) in OX40 knockout mice.

An important consideration for therapeutic intervention with OX40:Ig isto determine if memory is affected. During re-challenge with RSV orinfluenza, weight loss, recruitment of T cells and the frequency ofvirus-specific cytokine producing cells were unaffected in OX40:Igtreated mice. It is important to note that both fusion proteins wereadministered during the first infection and that their effect may bedifferent if administered during re-challenge.

The effect of OX40:Ig on virus induced illness and weight loss isstriking. In immunocompetent mice over-exuberant T cell responses andelevated TNF levels induce rapid and severe illness (Hussell, &Openshaw, J. Immunol. 165, 7109-7115 (2000); Hussell, et al, Eur JImmunol 31, 2566-2573 (2001). OX40:Ig reduced both CD4⁺ and CD8⁺ T cellsand weight loss during lung virus infection, reiterating theimmunopathological nature of the disease. Although a small (butsignificant) relative reduction of TNF producing CD8⁺ T cells wasobserved in OX40:Ig treated mice, this decrease is substantial when theactual numbers in the lung are calculated (mean control=2.1×10⁴, meanOX40:Ig=0.89×10⁴, p 0.01).

The reduction of both CD4⁺ and CD8⁺ T cells in OX40:Ig treated mice isconsistent with the expression of OX40 on both cell subsets. This is incontrast to a previous study in influenza or LCMV infected OX40 knockoutmice, where only the CD4⁺ T cells were affected²³. The discrepancy mayarise from differences between the mouse or virus strain used and/or thefact that our study used fusion proteins whereas Kopf et al. used OX40knockout mice. Similarly, intracellular IFNγ in CD4⁺ but not CD8⁺ Tcells was reduced in LCMV infected OX40 knockout mice but the sameanalysis was not performed for influenza virus. We show reducedintracellular IFNγ and TNF in both CD4⁺ and CD8⁺ T cells in mice treatedwith OX40:Ig, which is consistent with the decrease in IFNγ proteinproduction previously demonstrated in OX40 knockout mice (Kopf, et al.Immunity. 11, 699-708 (1999)). It is likely that the T cell requirementfor OX40 co-stimulation will alter with the infection model. Though CTLresponses to LCMV do not require CD4⁺ T cell help we do not know if thesame is true for influenza virus or RSV infection. Indeed, thymectomisedmice reconstituted with influenza immune splenic T cells that have beendepleted of CD4⁺ T cells, only generate half the normal level ofcytotoxic T cells after a subsequent virus challenge (Lightman, et alImmunology 62, 139-144 (1987)).

The reduced cellular infiltration in virus-infected mice treated withOX40:Ig can be explained in several ways, although the inventors do notwish to be bound by theory. OX40 ligation on the T cell has been shownto enhance primary T cell expansion, retention and anti-apoptosis geneexpression (Gramaglia, et al, J. Immunol. 165, 3043-3050 (2000);Gramaglia, et al, J. Immunol. 161, 6510-6517 (1998); Weinberg, et al,Semin. Immunol. 10, 471-480 (1998); Lightman, et al. Immunology 62,139-144 (1987)) but reduce peripheral T cell deletion (Maxwell, et al.J. Immunol. 164, 107-112 (2000)) and activation induced cell death(Weinberg, et al. Semin. Immunol. 10, 471-480 (1998)). OX40 ligand isalso expressed by endothelial cells (Imura, et al. J. Exp. Med. 183,2185-2195 (1996) and therefore OX40:Ig fusion protein treatment mayblock lymphocyte extravasation into the parenchyma Transgenic miceexpressing OX40 ligand under the control of the CD11c promoter haveincreased CD4⁺ T cells in B cell follicles (Brocker, et al. Eur. J.Immunol. 29, 1610-1616 (1999)). We show reduced cell recruitment to themediastinal lymph nodes by OX40:Ig treatment, which could representaltered extravasation or impaired T cell priming. Though OX40:OX40Linteractions influence T cell migration into the CNS during EAE (Nohara,et al. J. Immunol 166,2108-2115 (2001)) no defects in T cell homing havebeen detected in OX40L knockout mice (Chen, et al. Immunity. 11, 689-698(1999)).

Effective pulmonary clearance of C. neoformans requires T cell immunity(Huffnagle, et al. J.Exp.Med. 173, 793-800 (1991); Hill & Harmsen,J.Exp.Med. 173, 755-758 (1991)), monocytes and neutrophils (Mambula, etal. Infect. Immun. 68, 6257-6264 (2000)). Administration of OX40L:Igenhances anti-tumour immunity in vivo by increasing the number andsurvival of tumour specific T cells. We showed that CD4⁺ and CD8⁺ Tcells increased in the lung of C. neoformans infected mice treated withOX40L:Ig. In OX40L:Ig treated mice enhanced IFNγ secreting T cellscoincided with a reduction in fungal burden and eosinophilia in thelung. The converse treatment with OX40:Ig also reduced the number ofCD4⁺ T cells in the lung and hence the level of Th2 cytokines essentialfor eosinophil recruitment and survival. A similar effect on eosinophilsis also observed during ovalbumin aerosol exposure in OX40 knockout mice(Jember, et al. J.Exp.Med. 193, 387-392 (2001)). However, the kineticsof pathogen clearance with this fusion protein was not accelerated. Inthis model, therefore, OX40L:Ig treatment would be more beneficial thanOX40:Ig.

Many studies have attempted to explain Th1 or Th2 development based onthe level of co-stimulation received by the T cell including OX40signalling (Lenschow, et al, Annu. Rev. Immunol. 14, 233-58, (1996);Fowell, et al. Immunity. 6, 559-569 (1997); Gause, et al, J. Immunol.158,4082-4087 (1997)). We now report that inhibition of OX40co-stimulation interrupts both Th1 and Th2 responses during infectioninduced inflammatory disease. Treatment with OX40:Ig reduced IFNγ andTNF during lung viral infection and also Th2 dependent eosinophilic lungdisease in mice infected with C. neoformans. In addition OX40L:Igadministration enhanced IFNγ and IL-4 during RSV infection. Thissupports previous studies showing that T cell differentiation isprimarily dependent on the dose and affinity of peptide antigens ratherthan intrinsically reliant on co-stimulatory signals (Rogers & Croft. J.Immunol. 164, 2955-2963 (2000); Tanaka, et al. J Exp. Med 192, 405-412(2000)). Indeed OX40 ligand knockout mice show reduced Th1 and Th2responses to protein and alloantigens (Murata, et al. J. Exp. Med. 191,365-374 (2000)).

Our studies clearly show that harmful T cell immunity during lunginfluenza and RSV infection can be prevented by inhibiting OX40co-stimulation (using OX40:Ig fusion proteins); an effect that does notprevent viral clearance or recall responses and is also beneficial afterthe onset of clinical symptoms. More importantly we show for the firsttime that infection induced lung eosinophilia can be prevented by 1)promoting Th1 development (using OX40L:Ig fusion protein) and 2) byreducing CD4⁺ T cells (using OX40:Ig fusion protein). In the case ofOX40L:Ig administration this resulted in a reduced fungal burden.Manipulation of OX40 co-stimulation may therefore be beneficial forasthmatic and allergic conditions. Intervention of single co-stimulatorypathways may therefore be a realistic therapeutic strategy in theabsence of suitable vaccines.

Example 3

Methods

Mice and Pathogens

8-12 week old female C57BL/6 mice (Harlan Olac Ltd, Bicester, UK) werekept in pathogen-free conditions according to Home Office guidelines.IFN-γ^(−/−) and IL-12^(−/−) p40 mice (back-crossed to C57BL/6 backgroundat least 10 times) were purchased from The Jackson Laboratory (BarHarbor, Me.) and were maintained by homozygous matings under contract atBantin & Kingman Universal. All mice came from specific pathogen-freecolonies. C. neoformans strain 52, was obtained from the American TypeCulture Collection (Rockville, USA) and for infection grown tostationary phase (48-72 hours) at room temperature on a shaker inSabouraud dextrose broth (1% neopeptone and 2% dextrose; Difco, Detroit,Mich.). The cultures were washed in saline, counted on a haemocytometerand diluted in sterile nonpyrogenic saline to the required infectivedose.

Preparation of OX40 Ligand Fusion Proteins

Murine OX40L:murine IgG1 fusion protein (OX40L:Ig) was constructed asdescribed in Example 2 using a chimeric cDNA containing the C-terminalregion of OX40L fused to the constant region of murine IgG1. LPScontamination of fusion protein was analysed by gas chromotography-massspectrometry (GC-MS) of fatty acid methyl esters. Briefly, samples werederivatised using methanolic HCl and resulting fatty acid methyl esterswere dissolved in hexanes prior to on column injection on a Stabilwax(30m×0.25 mm internal diameter, Restek Corp., PA) column. Samples wereanalysed using a temperature gradient of 150° C. to 250° C. at a rate of3° C./min. Characteristic retention times and spectra of known standardswere used to identify any fatty acid methyl esters present in thesamples. All fusion protein used contained no LPS.

Mouse Infections and Treatment

On day 0, mice were anaesthetised with halothane and intranasallyinfected with 2×10⁴ cfu C. neoformans in 50 μl sterile PBS. Some groupsof mice were injected intraperitoneally (i.p.) with 100 μg OX40L:Igfusion proteins (Xenova pharmaceuticals, Cambridge, UK) or mouse IgG(Sigma-Aldric, Poole, Dorset) on various days after infection asindicated in the text Mice were then sacrificed at various time pointsafter C. neoformans infection by injection of 3 mg pentobarbitone andexsanguinated via the femoral vessels. Bronchoalveolar lavage fluid(BAL), lung tissue and sera were recovered using methods describedpreviously.

Cell Recovery

Briefly, the lungs of each mouse were inflated 6 times with 1 ml 1 mMEDTA in EMEM and placed in sterile tubes on ice. 100 μl BAL fluid fromeach mouse was cytocentrifuged onto glass slides. The remainder wascentrifuged and the supernatant removed and stored at −70° C. in 200 μlaliquots for analysis of cytokines by ELISA. Cell viability was assessedusing trypan blue exclusion and the pellet resuspended in RPMIcontaining 10% FCS, 2 mM/ml L-glutamine, 50 U/ml penicillin and 50 μg/mlstreptomycin (R10F) at a final concentration of 10⁶ cells/ml.Eosinophils were enumerated as granulocytes by flow cytometry, usingforward and side scatter. Identification was confirmed by countingeosinophils in H and E stained cytocentrifuge preparations.

Flow Cytometric Analysis of Intracellular and Cell Surface Antigens

1×10⁶ BAL and lung derived cells were stained using APC-conjugatedanti-CD4, anti-CD8-PerCP, anti-CD44-PE (all BD Pharmingen, Heidelberg,Germany) and anti-OX40-FITC (Serotec, Oxford, UK) for 30 min on ice.Cells were then fixed for 20 min at room temperature with 2%formaldehyde. To detect intracellular cytokines, 10⁶ cells/ml wereincubated with 50 ng/ml PMA (Sigma-Aldric, Poole, Dorset), 500 ng/mlionomycin (Calbiochem, Nottingham, UK), and 10 mg/ml brefeldin A(Sigma-Aldrich) for 4 hours at 37° C. Cells were then stained withanti-CD4-APC or anti-CD8-PerCP and fixed as described above. Afterpermeabilization with PBS containing 1% saponin/1% BSA/0.05% azide(saponin buffer) for 10 min FITC-conjugated anti-IFN-γ (Pharmingen) andPE-conjugated anti-IL5 (Pharmingen) diluted 1:50 in saponin buffer wereadded. 30 minutes later cells were washed once in saponin buffer andonce in PBS/1 % BSA/0.1% azide. All data was acquired on a FACSCaliburand analysed with CellQuest Pro software (BD Biosciences, Belgium). Toset limits of background fluorescence appropriately labelled isotypematched control antibodies were used.

Lung Histology

In some studies, lungs were inflated and fixed with 10% formalin in PBS,excised and embedded in paraffin wax by the histopathology department atthe Hammersmith Hospital, UK. 4 μm sections were stained withhaematoxylin and eosin (H and E). The lungs from 4-5 mice per group wereanalysed.

Enumeration of C. Neoformans from Lung Homogenates

Lungs were homogenised by passage through 100 μm cell strainers (BDlabware, New Jersey, USA). 100 μl of cell suspension was diluted in PBSand incubated at room temperature for 48 hours on sabouraud dextroseagar plates (Sigma). The total colony forming units per lung was thendetermined (number of colonies×dilution factor×original cell suspensionvolume).

Cryptococcus Neoformans-Specific Antibody ELISA

2×10⁵ cfu/ml heat killed C.neoformans in PBS was used to coat 96 wellmicrotiter plates overnight at room temperature on a shaker. Afterblocking with 3% BSA/PBS for 2 hours at 37° C., dilutions of sample serawere added for a further hour at room temperature. Bound antibody wasdetected using peroxidase-conjugated rabbit anti-mouse Ig andO-henylene-diamine as a substrate. The reaction was stopped with 50 μl2.5 M sulphuric acid. Optical densities were read at 490 nm and meanblank values (ODs from normal mouse serum) were subtracted from theoptical density values of test samples.

Immunohistochemical Determination of Cellular Proliferation

In vivo incorporation of BrdU (Sigma) was determined to assess cellularproliferation in the lung. Mice were injected intraperitoneally with 75mg/kg BrdU 90 minutes before sacrifice. Lungs were inflated with 10%paraformaldehyde in PBS and embedded in paraffin. 4-μm sections weredigested in 0.01% trypsin and then 1M hydrochloric acid. BrdUincorporation was detected with overnight incubation with a biotinlabelled anti-BrdU monoclonal antibody (Caltag laboratories, South SanFrancisco, Calif.) followed by streptavidin peroxidase (Dako Corp.,Carpinteria, Calif.) for 1 hour. Sections were then incubated with DABsubstrate (Vector laboratories inc., Burlingame, Calif.) for 10 minutes.To compare proliferation between groups, sections were analysedmicroscopically, and the percentage of positively stained cells wascounted in the perivascular regions in each sections. At least 400 cellswere counted per section.

Statistics

Statistical significance was evaluated using the student t test, 2tailed assuming unequal variance within the Minitab software program.

Results

C. Neoformans Induces OX40 Expressing T Cells in the Lung and Airway.

C57BL/6 mice infected with C. neoformans develop extensive pulmonaryeosinophilia that peaks around 11-14 days after infection. The kineticsof eosinophilia parallels that of C. neoformans cfu in the lung (FIG.15A). Peak recruitment of CD4⁺ T cells and to a lesser extent CD8⁺ Tcells also occurs at times of maximal eosinophilia and pathogen burden(FIG. 15B). As reported previously C57BL/6 mice do not completely clearthe infection and low levels remained in the lung indefinitely.

Surface expression of OX40 is up-regulated by T cells 1-2 days afterantigen stimulation. The uninfected mouse lung and airway did notcontain any CD4+ T cells expressing OX40. Of the CD4+ T cells recruitedto the lung during infection, however, up to 15% express OX40 (FIG.15C). Peak OX40 expression occurs first in the lung and then in theairway, which probably represents migration of T cells to the site ofhigh pathogen burden. A representative dot plot of OX40 expression onCD4⁺ T cells is shown in FIG. 15D. This typical staining pattern showsthat OX40 is expressed at various intensities on CD4⁺ T cells. Cellsnegative for CD4 but OX40+ are all CD8+ (data not shown).

OX40L:Ig Fusion Protein Enhances CD4+ T Cell Responses During C.Neoformans Infection.

After determining that OX40 was expressed by infiltrating T cells wethen determined whether T cell responses could be influenced by OX40L:Igfusion protein administration. In uninfected mice this fusion proteinhas no effect. Whilst not wishing to be bound by theory, we believe thisis due to i) the low number of T cells in naïve mice but moreimportantly ii) that those T cells present do not express OX40 untilactivated by antigen. In addition, administration of OX40L:Ig alone didnot induce any noticeable change in the cellular composition in the BALor lung (data not shown). Administration of OX40L:Ig to C. neoformansinfected mice, however, increased the kinetics of accumulation of Tcells in the lung (FIG. 16 a), which may represent increased survivaland/or proliferation. Enhanced inflammatory infiltration can clearly beobserved in H and E stained sections of lung. In untreated mice aminimal infiltrate is observed around the airways and blood vessels 11days post-infection (FIG. 16 b). OX40L:Ig treatment significantlyincreases this inflammatory infiltration (FIG. 16 c).

To determine the effect on cell proliferation BrdU was administrated 90minutes before termination of OX40L:Ig and control treated C. neoformansinfected mice. Lungs were then inflation fixed, removed, embedded inparaffin and 4 μm sections stained with an anti-BrdU antibody. For 10days after C. neoformans infection there was no discernible differencein the number of lung cells expressing BrdU. By day 11 however therewere significantly more proliferating cells in the lungs of OX40L:Ig(FIG. 17 b) compared to control (FIG. 17 a) treated mice (FIG. 17 c).This difference persisted throughout the remainder of the time course.

OX40L:Ig Controls Pathogen Burden and Reduces Associated Eosinophilia.

Since T cell numbers were increased we next determined the effect ofOX40L:Ig fusion protein treatment on pathogen burden and associatedeosinophilia. Similar colony forming units were recovered from infectedmice during initial time points regardless of the treatment. From day 12onwards, a significantly reduced pathogen burden was observed inOX40L:Ig treated mice compared to controls in three independentexperiments (FIG. 18 a). The recruitment of eosinophils to the lungfollows similar kinetics to that of the pathogen (i.e. both are minimalat early stages after infection and peak around days 12-15). OX40L:Igtreatment reduced the percent eosinophilia in the lung (FIG. 18 b).

OX40L:Ig Treatment Increases IFN-γ in CD4⁺ T Cells.

Since eosinophilia in this model requires CD4⁺ T cells secreting type 2cytokines, we next investigated the cytokine profile in treated mice byintracellular cytokine staining. Only a few lung CD4⁺ T cells expressedintracellular IFN-γ in control treated mice (FIG. 19 a). OX40L:Ighowever increased this relative production 2-3 fold (FIG. 19 b) andtotal numbers were increased from 1.61±1.34×10⁴ cells in control mice to11.67±3.4×10⁴ cells in OX40L:Ig treated mice (p<0.05). By calculatingthe ratio of IFN-γ:IL-5 expressing cells we can see that OX40L:Igtreatment significantly shifts the CD4⁺ T cell cytokines in the lung toa Th1 phenotype (FIG. 19 c). Consistent with this we observed areduction in IgE 7 days post-infection in nasal wash following OX40L:Igtreatment compared with control treated mice (4/5 mice, p<0.05).

IFN-γ is Required for the Reduction in Pathogen Burden and Eosinophiliaby OX40L:Ig

The mechanism by which OX40L:Ig mediates its effect was theninvestigated in IFN-γ and IL-12 knockout mice. Wild typeimmuno-competent mice produced similar results to previous experimentsby showing increased CD4⁺ T cells FIG. 20 a) but reduced pathogen burden(FIG. 20 b) and eosinophils (FIG. 20 c) after OX40L:Ig treatment.Experiments in the knockout mice produced surprising results. Theabsence of IFN-γ or IL-12 did not affect the OX40L:Ig induced incrementin CD4+ T cell numbers (FIG. 20 a) or their activation (data not shown).The ability of OX40L:Ig to reduce pathogen burden (FIG. 20 b) andeosinophilia (FIG. 20 c) however was impaired and indistinguishable fromcontrol treated wild type groups. We originally thought that eosinophilsmight actually increase in both knockout mouse strains due to impairedIFN-γ production. This was not the case despite the low levels of IFN-γand the small but significant increase in intracellular IL-5 productionby CD4⁺ T cells in IL-12 knockout mice (Table 1). Total CD4/ Total CD4/Strain Treatment % CD4/IFN-γ IFN-γ (×10⁻⁴) % CD4/IL-5 IL-5 (×10⁻⁴)IFN-γ^(−/−) Control Ig 0.105 ± 0.03 ± 3.1 ± 1.3 ± 0.137 0.03 0.29 .055OX40L:Ig 0.15 ± 0.008 ± 9.48 ± 2.35 ± 0.3 0.015 8.22 2.96 IL-12^(−/−)Control Ig 1.36 ± 1.09 ± 5.9 ± 5.33 ± 0.74 0.34 2.65 2.57 OX40L:Ig 3.6 ±3.32 ± 12.9 ± 14.73 ± 1.26* 1.62* 3.67* 10.74Discussion

The OX40:OX40 ligand axis presents a novel method of targeting activatedT cells since OX40 is only induced after T cells receive two initialsignals: TCR interaction with MHC class I or II bound peptide and theCD28:B7 interaction. There are a number of infectious diseases whereprotection is poor due to insufficient or transient immunity. Thespecific enhancement of responding T cells at the time of infection orvaccination in situ may therefore afford better protective immunitywithout disturbing the rest of the T cell repertoire. Current immuneadjuvants enhance immune responses by increasing antigen uptake or thekinetics of inflammatory reactions. This can have unwanted side effectsincluding immune recognition of the adjuvant and toxicity. Similarlyimmune reactions could be promoted using B7 fusion proteins, but thiswould affect naïve as well as antigen activated T cells. We now reportfor the first time the beneficial effect of promoting T cells via OX40engagement during an infectious disease using OX40 ligand fusion protein(OX40L:Ig). During C. neoformans infection OX40L:Ig administrationpromotes cell mediated immunity and IFN-γ production by CD4⁺ T cells,while reducing the pathogen burden in the lung and the associatedeosinophilia. Such fusion proteins also have a longer half life(approximately 4 days) compared to antibody (17 hours) and can beconstructed entirely of human proteins without the necessity for“humanisation” or more complex CDR3 grafting.

OX40L:Ig administration alone does not produce any visible effect innaïve mice and yet has a profound influence when present duringinflammation caused by C. neoformans. The critical trigger appears to bethe level of OX40 expressed by the T cell, which is virtually absent inuninfected mice but increases on CD4⁺ T cells during infection. The nextreasonable question to pose during C. neoformans infection wouldtherefore be why doesn't the endogenous natural interaction between OX40and OX40 ligand cause the same effect in the absence of the fusionprotein. OX40 ligand expression, though constitutive on cardiac myocytesand a subset of DCs in SCID mice is limiting in the absence of a “dangersignal”. This signal can be provided by a strong adjuvant, such ascomplete Freunds adjuvant or LPS. Studies detailing the ability ofinfections to provide a sufficient OX40 ligand inducing signal to theAPC are limiting. We show however that C. neoformans is equivalent toLPS in its ability to induce OX40 ligand. The beneficial effect offusion protein administration is therefore likely to be the provision ofincreased engagement over and above that naturally provided in vivo.This hypothesis is supported by studies showing a three-fold increase ingerminal centre T cells in transgenic mice over-expressing OX40 ligandin dendritic cells. Similarly, stimulation of OX40 using anti-OX40antibody increases T cells specific for soluble, or super, antigens.Combined with LPS and a 60 fold increase is observed. An alternativeexplanation is that enhanced signals via OX40 using OX40L:Ig overridesthe negative signals provided by CTLA-4. This hypothesis is importantsince C. neoformans induces T cell expression of CTLA-4 and anti-CTLA-4treatment has been shown to enhance pathogen clearance.

The increase of CD4⁺ T cells by OX40L:Ig in our study is likely toreflect enhanced proliferation and/or survival due to the reportedup-regulation of survival factors Bcl-2 and Bcl-X_(L). Enhanced BrdUuptake by infiltrating cells was clearly observed in lung sections. Itis interesting to note that this effect was only observed 12 days afterC. neoformans infection. This time point is consistently associated withmaximal pathogen burden and eosinophilia There is therefore a lag phaseduring which T cells are recruited and antigen-activated before OX40L:Igcan exert its effect. Enhanced T cell responses by OX40 engagement havealso been reported in TCR transgenic mice where responsiveness toantigen remained 95 days after initial antigen exposure and in vitrousing OX40L transfected fibroblasts.

One concern during our studies was that OX40L:Ig would preventextravasation of T cells into the lung since the ligand is alsoexpressed by endothelial cells and mediates adhesion of activated Tcells. If T cells expressed OX40 prior to exiting the blood then OX40:Igmay bind and prevent interaction with the native ligand on endothelialcells. The increment in T cell numbers in the lung suggests that thisdid not occur. This is probably due to the lack of OX40 expression on Tcells prior to antigen activation in the lung itself.

The eosinophilic response during C. neoformans infection is dependent onT cells secreting type II cytokines. The reduction of eosinophils byOX40L:Ig treatment may therefore be a consequence of one or more of thefollowing: 1) enhanced direct apoptosis by IFN-γ, 2) reduced IL-5 or 3)decreased T cell stimulation due to the lower pathogen burden. Whilstnot wishing to be bound by theory, we believe option one is more likelysince IFN-γ was increased 3 fold, T cell numbers were actually increasedand eosinophils were not reduced in IFN-γ or IL-12 knockout miceinfected with C. neoformans and treated with OX40L:Ig. Though we did notobserve a change in intracellular IL-5 expression in CD4+ T cells thecytokine balance is definitely in favour of a Th1 environment. Theinduction of IFN-γ by enhanced OX40 signalling has been describedpreviously in re-call responses to antigens encountered during OX-40engagement. CD8⁺ intraepithelial lymphocytes expressing OX40 and OX40Lafter CD3 stimulation also produce high levels of IFN-γ. The cytokineretort by T cells after engagement of OX40 appears to alter depending onthe cell population and the antigen being studied since other data showseffects on Th1 and/or Th2 cytokines.

The reduction of C. neoformans by OX40L:Ig is also likely to bedependent on enhanced IFN-γ, which activates macrophages and increasestheir fungicidal activity. CD4⁺ T cells play an important role inrecruiting macrophages during virulent Cryptococcal infection. In ourstudy we observed increased CD4⁺ T cells secreting IFN-γ and an increasein the number of macrophages (data not shown). This therefore providesan environment more equipped to manage fungal clearance. This idea issupported by the inability of OX40L:Ig to decrease cfus in IL-12 andIFN-γ knockout mice. CD4⁺ T cells expressing OX40 are also present inthe lung earlier during OX40L:Ig treatment and may therefore assistmacrophages in the early control of the pathogen. Previous studies havedetailed the importance of IL-12 in protection from C. neoformans.Optimal production of IL-12 requires CD40 on T cells interacting withCD40L on APCs. Blockade of this interaction decreases IFN-γ andfungicidal activity. The presence of more T cells in OX40L:Ig treatedmice may therefore enhance CD40:CD40L interactions.

Since we have demonstrated an alteration in the cytokine environment thenext question was what has happened to antibody production? SinceOX40:OX40L interactions are thought to play a role in antibodyproduction, we initially thought that the OX40L:Ig treatment may blockthe signal to OX40L expressing B cells. Total B cell numbers and IgEproduction in the BAL were not affected (data not shown) in OX40L:Igtreated mice, presumably due to the presence of other B cell stimulatorysignals such as CD40 and CD70. This is similar to the absence of anyaffect on B cell immunity reported in OX40 knockout mice.

It is interesting to note that OX40L:Ig still increased T cell numbersin both IL-12 and IFN-γ knockout mice. Such cytokine defects do notappear to prevent antigen presentation and T cell clonal expansion.IL-12 in addition to increasing IFN-γ is also reported to induce TNF-α,GM-CSF, IL-8 and even IL-10. These do not however appear to compensatefor a lack of IFN-γ in knockout mice.

In summary, this example shows for the first time that OX40 ligation canreduce the symptoms of a lung infection. This effect was dependent onIFN-γ and IL-12. Since OX40 is expressed on antigen activated T cellsthis represents a novel strategy to enhance immunity to infectiousdiseases. Specific enhancement in the number and function of T cellsactually participating in an immune response has not previously beentested in an infectious disease. OX40L:Ig fusion proteins have beenshown to enhance tumour immunity. This strategy therefore holdssignificant promise for the plethora of infections that induceinsufficient immunity or latency.

The invention is susceptible of modifications and variations as will beapparent to those skilled in the art, and the present disclosure extendsto combinations and subcombinations of the features mentioned ordescribed herein and in the published documents referred to, which areincorporated by reference in their entirety for all purposes.

1. A pharmaceutical composition comprising one or more compounds thatinhibit immune system costimulation by OX40.
 2. The method of claim 18,wherein the disease is inflammation of the lung.
 3. The method of claim18, wherein the disease is caused by influenza virus.
 4. The method ofclaim 18, wherein the disease is caused by respiratory syncytial virus(RSV).
 5. The method of claim 18, wherein the disease is caused bybacterial infection.
 6. The method of claim 5, wherein the bacterialinfection is caused by C. neoformans.
 7. A compound that inhibits immunesystem costimulation by OX40 comprising a fusion protein comprising anOX40 domain and an immunoglobulin domain.
 8. The compound of claim 7,wherein the fusion protein comprises the extracellular domain of OX40linked via its C-terminus to the N-terminus of the constant domain ofthe immunoglobulin.
 9. The method of claim 20, wherein the OX40 andimmunoglobulin are from the same species as the subject to be treated.10. A method for the treatment, amelioration or prevention of diseasesassociated with virus, bacteria or other infective agent, comprisingadministering to a subject one or more compounds that can inhibit immunesystem costimulation by OX40.
 11. (canceled)
 12. A product containingone or more compounds that can inhibit immune system costimulation byOX40; and an anti-inflammatory drug as a combined preparation forsimultaneous, sequential or separate use in treating, ameliorating orpreventing diseases associated with virus, bacteria or other infection.13. (canceled)
 14. A composition comprising a vaccine and one or morecompounds that can inhibit immune system costimulation by OX40.
 15. Acomposition as claimed in claim 14, wherein the vaccine is an anti-viralor anti-bacterial vaccine.
 16. (canceled)
 17. A product containing ananti-viral vaccine and one or more compounds that can inhibit immunesystem costimulation by OX40 as a combined preparation for simultaneous,sequential or separate use in vaccination against said virus.
 18. Amethod of treating a subject for a disease associated with an infectiousagent selected from the group consisting of: virus, bacteria and otherinfective agent comprising administering the pharmaceutical compositionof claim 1 to the subject.
 19. The composition of claim 7, wherein theimmunoglobulin is IgG.
 20. A method of treating a subject for a diseaseassociated with an infectious agent selected from the group consistingof: virus, bacteria and other infective agent comprising administeringthe pharmaceutical composition comprising the compound of claim 7 to thesubject.